parkin structure and function

STATE-OF-THE-ART REVIEW

Parkin structure and functionMarjan Seirafi, Guennadi Kozlov and Kalle Gehring

Department of Biochemistry and the Groupe de Recherche ax�e sur la Structure des Prot�eines, McGill University, Montr�eal, Qu�ebec, Canada

Keywords

mitophagy; parkin; PINK1; ubiquitin;

ubiquitination

Correspondence

K. Gehring, Department of Biochemistry,

McGill University, 3649 Promenade Sir

William Osler, Montr�eal, Qu�ebec, Canada

H3G 0B1

Fax: 514 398-2983

Tel: 514 398-7287

E-mail: [email protected]

(Received 4 October 2014, revised 5

January 2015, accepted 24 February 2015)

doi:10.1111/febs.13249

Mutations in the parkin or PINK1 genes are the leading cause of the auto-

somal recessive form of Parkinson’s disease. The gene products, the E3

ubiquitin ligase parkin and the serine/threonine kinase PINK1, are neuro-

protective proteins, which act together in a mitochondrial quality control

pathway. Here, we review the structure of parkin and mechanisms of its

autoinhibition and function as a ubiquitin ligase. We present a model for

the recruitment and activation of parkin as a key regulatory step in the

clearance of depolarized or damaged mitochondria by autophagy (mito-

phagy). We conclude with a brief overview of other functions of parkin

and considerations for drug discovery in the mitochondrial quality control

pathway.

Introduction

Parkinson’s disease (PD) is a degenerative movement

disorder caused by the progressive death of dopamine-

producing neurons in the substantia nigra pars com-

pacta of the mid-brain. Studies linking PD to defects

in the electron transport chain suggest that damaged

mitochondria may play a central role in PD pathology

[1]. While most PD cases occur sporadically, research

on inherited forms of PD in the past decade have shed

new light on the disease and pathogenesis. Studies of

two recessive PD genes, PINK1 (PTEN-induced puta-

tive kinase protein 1 or PARK6) and parkin

(PARK2), have provided direct evidence for the con-

tribution of damaged mitochondria in PD pathology

[2]. Parkin is a cytosolic E3 ubiquitin ligase and

PINK1 is the only known protein kinase with a

mitochondrial targeting domain. These two proteins

are involved in a common pathway regulating

mitochondrial quality control and promoting the selec-

tive autophagy of depolarized mitochondria (mito-

phagy) [3]. Under basal conditions, parkin E3 ligase

activity is repressed in the cytoplasm and PINK1 is

imported into mitochondria via TOM40 and TOM20

core containing complexes [4] and cleaved sequentially

by mitochondrial proteases such as MPP and PARL

[5–8]. Cleaved PINK1 then degrades rapidly in the

cytosol via the N-end rule pathway so that levels of

PINK1 are low in healthy cells [9]. However, when the

organelle loses its inner membrane electrochemical gra-

dient, import of PINK1 to the mitochondria is inhib-

ited and the protein is stabilized on the mitochondrial

outer membrane with its kinase domain on the cyto-

solic face [10,11]. The accumulation of PINK1 kinase

activity on the mitochondria triggers parkin recruit-

ment and activation. Activated parkin produces

Abbreviations

CCCP, carbonyl cyanide m-chlorophenyl hydrazone; HECT, homologous to the E6-AP carboxyl terminus; IBR, in-between-RING; PD,

Parkinson’s disease; RBR, RING-between-RING; REP, repressor element of parkin; RING, really interesting new gene; ROS, reactive oxygen

species; Ubl, ubiquitin-like domain; UIM, ubiquitin interacting motif.

2076 FEBS Journal 282 (2015) 2076–2088 ª 2015 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and

distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

http://creativecommons.org/licenses/by-nc-nd/4.0/

ubiquitin chains on various outer mitochondrial

membrane proteins leading to autophagic elimination

of the damaged organelle [12–15]. Pathogenic muta-

tions in either of these genes lead to loss of this quality

control pathway and accumulation of impaired

mitochondria, which are thought to be a source of

toxic reactive oxygen species (ROS) and contribute to

neuronal cell death and PD.

Parkin is an RBR E3 ubiquitin ligase

Ubiquitination is a post-translational modification that

typically marks proteins for degradation through the

covalent attachment of ubiquitin and ubiquitin chains

to lysine residues or the N-terminal amino group of a

substrate protein [16]. In addition to degradation via

the proteasome, ubiquitination can act as a signal for

autophagy [17] – degradation via lysosomes – as well

as alter substrate protein activity or location [18].

Ubiquitination is carried out through the sequential

action of three enzymes: E1 ubiquitin-activating

enzymes, E2 ubiquitin-conjugating enzymes and E3

ubiquitin ligases (Fig. 1A). In the pathway, E1 first

uses ATP to activate ubiquitin for conjugation by

forming a thioester between its catalytic cysteine and

the C-terminal carboxyl group of ubiquitin. The

ubiquitin is then passed to a second cysteine of an E2

ubiquitin conjugating enzyme. In the final step, the

ubiquitin-charged E2 enzyme interacts with a specific

E3 ubiquitin ligase and transfers the ubiquitin to the

amino group of a substrate protein. Ubiquitin contains

seven lysine residues as well as an N-terminal amino

group that can be used to build chains of polyubiqu-

itin. The most common chain types are K48 and K63

chains in which multiple ubiquitin molecules are linked

in a linear arrangement with the C terminus of one

molecule attached to lysine 48 (or 63) of the next. In

the ubiquitination pathway, the E3 ubiquitin ligases

typically provide the majority of the specificity and

regulation in recognition of substrates and control of

activity [19]. Based on considerations of structure and

chemistry, three classes of E3 ligases are distinguished:

RING-type (including U-box ligases), HECT-type and

RING-HECT hybrids [20] (Fig. 1B). RING-type E3s

are characterized by the presence of a canonical

C3HC4-type RING (really interesting new gene)

domain that binds the E2 enzyme but does not partici-

pate directly in catalysis. This class of E3s functions as

inert scaffolding ligases that facilitate the direct trans-

fer of ubiquitin from the E2 onto the substrate.

HECT-type E3s contain a HECT (homologous to the

E6-AP carboxyl terminus) domain with an active site

cysteine, which accepts ubiquitin from an E2 enzyme

in the form of a thioester intermediate and then trans-

fers it to substrates. The third class consists of the

RBR (RING-between-RING) family of E3 ubiquitin

ligases that combine the chemistry of HECT-type lig-

ases with structural similarity to RING-type ligases

[21,22]. RBRs contain a canonical C3HC4-type RING

(named RING1), followed by two conserved Cys/His-

rich Zn-binding domains, in-between-RING (IBR) and

RING2 domains, that contain an active site cysteine

residue. This cysteine accepts ubiquitin from the E2

enzyme and transfers it onto substrates; hence, ligases

in this class are sometimes referred to as RING-HECT

hybrids [22].

Parkin, a member of RBR E3 ubiquitin ligases,

ubiquitinates a wide variety of cytosolic and outer

mitochondrial membrane proteins upon mitochondrial

depolarization [23,24]. It forms multiple types of

ubiquitin chains, most frequently K63, K48, K11 and

K6 linkages [25]. Parkin also shows relatively lax sub-

strate specificity [23,24]. Accumulation of polyubiqu-

itin chains on mitochondria signals recruitment of the

autophagosome and proteasome machinery to initiate

mitophagy [12–14,23]. Parkin itself becomes ubiquiti-

nated by the attachment of K6 ubiquitin chains, which

may play a role in its own degradation [26]. The activ-

ity of parkin is tightly regulated and normally

repressed [27–30]. In cells, parkin activity can be acti-

vated under a variety of conditions such as depolariza-

tion of mitochondria or epidermal growth factor

signaling [31]. A large number of treatments that dis-

rupt its structure are also known to de-repress its

ligase activity in vitro. These include heat treatment,

N-terminal deletions and some point mutations [28–30,32].

Lessons from the structure

Recent structures of parkin have revealed much about

its regulation and function (Fig. 2A). Parkin consists

of a ubiquitin-like domain (Ubl) at its N terminus and

four zinc-coordinating RING-like domains: RING0,

RING1, IBR and RING2. More than 120 pathogenic

PD mutations are spread throughout parkin domains,

attesting to critical functions for each of the individual

domains [33]. The Ubl domain is involved in substrate

recognition, binding SH3 and ubiquitin interacting

motif (UIM) domains, proteasome association, and

regulation of cellular parkin levels and activity

[27,31,34–37].Last year, several groups reported high-resolution

crystal structures of a parkin fragment consisting of

the RING0, RING1, IBR and RING2 domains (PDB:

4K7D, 4I1H, 4I1F, 4BM9) along with a low-resolution

2077FEBS Journal 282 (2015) 2076–2088 ª 2015 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

M. Seirafi et al. Parkin structure and function

http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4K7D

http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4I1H

http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4I1F

http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BM9

structure of the full-length protein (PDB: 4K95) [28–30,38]. In the structures, parkin adopts a compact

arrangement, stabilized by multiple hydrophobic inter-

actions, similar to a coiled snake [39] (Fig. 2B). The

N-terminal Ubl domain uses a hydrophobic surface

centered around Ile44 to bind to the RING1 domain,

while the C-terminal catalytic domain is tightly associ-

ated with the RING0 domain. Domains RING0

through RING2 (collectively referred to as R0–RBR)

each coordinate two zinc ions through histidine and

cysteine residues, confirming the stoichiometry of eight

zinc ions per parkin [40]. The RING0 domain binds

zinc ions in a hairpin arrangement unique to parkin

while the RING2 and IBR domains show a sequential

arrangement of zinc-coordinating residues. RING0

and RING2 were originally identified as RING

domains based on the primary amino acid sequence

but their structural topology differs from a classical

RING fold. The RING1 domain is the only RING

domain with the cross-brace zinc coordination topol-

ogy observed in other RING-type E3 ligases. The simi-

larity of RING1 to other RING E3 ligases suggested

that it is the E2 binding site on parkin (Fig. 2C).

NMR titrations, mutagenesis and molecular modeling

ATP

E1

S

UbCO

UbCO

O-

Substrate

E2

S

UbCO

E3

Substrate

E2

S

UbCO

E2

SHE1

SH

E3

Substrate

NH

UbCO Ub Ub Ub

Substrate

NH

UbCO

UbUb

Ub

UbUb

UbUb

Substrate

NH

UbCO

Mono-ubiquitinationEndocytosis

K48 polyubiquitinationProteosomal & lysosomal degradation

Substrate

NH

CO

Linear chain polyubiquitinationNF-kB signalling

K63 polyubiquitinationSignalling, DNA repair

Lysosomal degradation

NH2

Substrate

E2

S

UbCO

RING

Substrate

E2

S

UbCO

NH2

RING

HECT E3E2

S

UbCO SH

E2

S

UbCO

SH

RINGE3

N-lope

C-lope

N-lope

C-lopeSubstrateSubstrate

NH2

RING1 IBR RING2RING/HECT hybrid

E3Substrate

RING1 IBR RING2 Substrate

NH2

A

B

Fig. 1. Ubiquitination pathway. (A) Cascade of ubiquitination enzymes. The ubiquitin-activating enzyme E1 uses ATP to conjugate the C-

terminal carboxylic acid group of ubiquitin to an active site cysteine. This is then transferred to a cysteine on one of a number of E2

enzymes that work in concert with E3 enzymes to ubiquitinate substrate proteins on amino groups of lysine residues or protein N termini.

The formations of mono-ubiquitin or polyubiquitin chains on substrates are signals for different downstream pathways. (B) Classes of E3

ubiquitin ligases. E3 ligases can be distinguished based on the presence of a RING domain, a catalytic cysteine or both. RING and U-box

domain ligases act as scaffolds to bring the substrate and ubiquitin-conjugated E2 together. HECT ligases have a catalytic cysteine in their

C-lobe that is transiently conjugated to ubiquitin as an intermediate in a two-step process of substrate ubiquitination. Parkin is a RING/HECT

hybrid ligase that contains a RING domain that binds the E2 enzyme and a catalytic cysteine that transfers ubiquitin to the substrate.


Parkin structure and function M. Seirafi et al.

http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4K95

confirmed this and identified an a-helix (residues 263–271) and two loops (residues 239–244 and 290–292) inRING1 as the E2 binding site [28–30]. The RING2

domain is the catalytic module harboring the catalytic

cysteine (Cys431). While RING0 is unique to parkin,

the IBR domain is conserved among the RBR E3 fam-

ily; the precise function of the domains is currently

unknown. Parkin contains two interdomain linkers

that are flexible and not observed in the crystal struc-

tures. The first consists of 70 poorly conserved residues

of unknown function that follow the Ubl domain. The

second, within the R0–RBR fragment, occurs between

the IBR and RING2 domains and is composed of dis-

ordered segments and a conserved a-helix (residues

391–403) that binds to the RING1 domain. This helix

has been termed the repressor element of parkin

(REP) due to its role in the regulation of parkin activ-

ity.

Parkin is a difficult protein to work with unless cer-

tain precautions are taken [41]. The ligase activity is

normally repressed and surprisingly some mutations or

heat treatment increase activity [27–30]. The bound

zinc ions are required for structural stability and prop-

erly regulated enzymatic activity [40,42]. While supple-

mental zinc is not required during purification, metal-

chelating agents such as ethylenediaminetetraacetic

acid (EDTA) need to be avoided since they denature

the protein. Unfortunately, some published co-immu-

noprecipitation experiments have used EDTA and

need to be interpreted with caution. With a total of 35

cysteine residues, parkin requires a high concentration

of a reducing agent such as dithiothreitol to maintain

the cysteine thiols reduced and available for coordinat-

ing zinc ions.

The parkin structure provides rationales for many

of the mutations associated with PD (Fig. 2A). Certain

mutations compromise the structural integrity of the

protein. Others interfere with binding of substrates or

directly affect the enzyme catalysis. Among the best

understood, mutations C212Y, C289G and C441R

affect zinc coordination, while R42P, K211N and

T351P disrupt protein folding or stability. C431F and

G430D are in the catalytic site, and T240R prevents

E2 binding. The effects of other mutations such as

R33Q, D280N, G328E or T415N are less clear. These

may disrupt the interdomain interactions or affect

IBR

E2 enzyme

RING0

RING2

RING1

Cys431

Cysteine

R33Q R42P V65E K161N

M192L C212Y R234QT240M

R275WD280N

C289GG328E

R334CT351P T415N

G430DC431F

E444QW453s top

Ubl RING0 RING1 IBR RING2

1 76 141 225 327 378 410 465

REP

A

A398T

IBR

RING0

RING2

RING1

REPUbl

Phospho-siteSer65

Catalytic siteCys431

B C

K211N

Fig. 2. The structure of parkin reveals the mechanism of its autoinhibition. (A) Domain architecture of the five parkin domains and

identification of selected PD mutations. (B) Cartoon representation of parkin (PDB: 4K95) [29]. Parkin activity depends on two functional

sites: a binding site for ubiquitin-conjugated E2 enzyme on the RING1 of parkin and a catalytic site with a cysteine that forms a transient

covalent linkage with ubiquitin on the RING2 domain. Both sites are occluded in the autoinhibited structure. The Ubl domain and REP linker

between the IBR and RING2 domains prevent the E2 from binding to RING1. The RING0 domain partially covers the catalytic cysteine on

the RING2 domain. Mutations at interdomain interfaces as well as phosphorylation of Ser65 in the Ubl domain increase parkin ubiquitin

ligase activity. The eight structural zinc ions in parkin are shown as gray spheres. Dashed lines indicate portions of the IBR and RING2 linker

that were not observed in the crystal structure. (C) Model of an E2 enzyme bound to parkin with Ubl and REP linker removed. An additional

structural rearrangement must occur to allow ubiquitin on the E2 enzyme to transfer to the catalytic cysteine of parkin. The E2 and parkin

catalytic cysteines are ~ 50 �A apart in the model.



http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4K95

binding of other proteins by parkin. A comprehensive

list of known disease-causing parkin mutations

together with the predicted structure rationale and bio-

chemical effects is provided as supplementary material

by Wauer and Komander [30].

Autoinhibition

The activity of parkin is tightly controlled by multiple

mechanisms of autoinhibition. The first is access to the

catalytic RING2 domain, which is blocked by RING0.

Other RBR E3 ligases show similar mechanisms of au-

toinhibition [43–45]. The Ariadne domain of the RBR

E3 HHARI and the UBA domain of the RBR E3

HOIP both participate in inhibiting the catalytic

domain. Deletion of the N terminus of parkin through

to the RING0 domain leads to very high ubiquitin

ligase activity in vitro [28–30].A second mechanism is the control of binding of the

upstream E2 enzyme to parkin. Modeling and muta-

genesis have confirmed that the E2 binding site is on

the RING1 domain but the site is occluded by the Ubl

domain and REP linker (Fig. 2C). Deletion of the Ubl

domain and mutagenesis of the REP linker both

increase the affinity of E2 binding and parkin activity

[27,29]. Finally, the large distance between the E2

binding site and catalytic site on RING2 prevents

transfer of ubiquitin from the ubiquitin–E2 conjugate

to the parkin catalytic cysteine (Fig. 2C).

Catalytic mechanism

The detailed structure of parkin RING2 domain

reveals conserved chemistry across the RBR family of

E3 ligases for ubiquitin transfer to the target protein

(Fig. 3). Catalytically, ubiquitination proceeds

through two steps: (a) formation of a ubiquitin–cyste-ine thioester intermediate where the C terminus of

ubiquitin is covalently linked to the parkin catalytic

cysteine, and (b) acyl transfer where the ubiquitin is

transferred from parkin to an amino group of the

substrate.

A C

B

Fig. 3. Mechanism of parkin ubiquitin ligase activity. (A) Sequence alignment of RBR E3 ubiquitin ligases shows conservation in the catalytic

RING2 domains. The catalytic cysteine (highlighted in yellow) is invariant across RBR proteins, whereas histidine (green) and glutamate

residues (red) that play secondary roles in catalysis are less conserved. Gray indicates zinc-coordinating residues. (B) Structural comparison

of catalytic domains of parkin, HHARI and HOIP (PDB: 4K7D [29]; 4KBL [43]; 4LJO [44]). The alignment of the catalytic cysteine, histidine

and glutamate/glutamine is suggestive of a catalytic triad where the histidine acts as a general base to promote transesterification of

ubiquitin onto the substrate. (C) Model of the positions of the donor and acceptor ubiquitin molecules in the catalytic site of parkin based on

the crystal structure of HOIP [44]. The C terminus of the donor ubiquitin lies in a groove of the RING2 domain and terminates next to the

catalytic cysteine, mimicking the thioester intermediate. The amino group of the acceptor ubiquitin approaches Cys431 from the opposite

side but would be sterically blocked by the parkin RING0 domain in the absence of a conformational change.



http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4K7D

http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4KBL

http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4LJO

The catalytic cysteine residue (in the RING2

domain) is conserved in all the members of the RBR

family as are the residues involved in zinc coordina-

tion. Following the cysteine, there is partial conserva-

tion of a histidine residue and an acidic residue. In

parkin, these residues adopt a linear arrangement that

has been proposed to act as a catalytic triad where the

histidine acts as a general base to promote catalysis

[28–30]. The structures of two other RBR ligases are

known: HHARI, a member of the Ariadne family of

E3 ligases [43], and HOIP, a component of the linear

ubiquitination assembly complex (LUBAC) [44]. The

RING2 domains are highly conserved and share the

linear arrangement of the three residues (Fig. 3B).

Nonetheless, the results of mutagenesis are ambiguous

about the importance of the catalytic triad. The effect

of the loss of the histidine is substrate dependent and

can be suppressed in vitro by raising the pH [28,44]. In

cultured cells, mutation of the histidine only moder-

ately slows parkin-mediated mitophagy. And, while

the mutation E444Q reduces autoubiquitination [29]

and may be implicated in PD [46], it has no effect on

parkin activity in cells [28].

Additional insight into the catalytic mechanism

comes from the crystal structure of HOIP, which con-

tains two ubiquitin molecules in contact with catalytic

RING2 domain (Fig. 3C). The C terminus of one

ubiquitin molecule is positioned to mimic the thioester

linkage with the catalytic cysteine while the amino

group of the second ubiquitin molecule approaches the

cysteine from the other side and occupies the position

of the acceptor molecule. Transposition of the two

ubiquitin molecules onto the parkin crystal structure

generates a hypothetical model of the active site with a

thioester intermediate prior to acyl transfer. While the

donor ubiquitin can be accommodated to fit in a

groove on the surface of the RING2 domain, the posi-

tion of the acceptor ubiquitin or substrate protein

clashes with the RING0 domain, implying that this

domain must move in order for a substrate amino

group to access the active site.

Parkin activation

Recent studies have made tremendous progress in

understanding how parkin is activated. In vitro studies

of parkin have found that a wide variety of effectors

can promote parkin activity. These include point

mutations that disrupt inhibitory interdomain interac-

tions as well as N-terminal deletions [27–30]. The Ubl

domain plays a special role in parkin activation and a

number of binding partners that bind the Ubl domain

are associated with parkin activation [27]. The Ubl

domain recruits these binding partners such as the

SH3 domain and UIMs of endophilin A1, Eps15, pro-

teasomal subunits and ataxin-3 [31,35–37] through the

same hydrophobic surface centered around Ile44 that

it uses to interact with the RING1 domain, indicating

that the Ubl dissociates from RING1 upon activation.

PINK1 acts upstream of parkin and is required for

parkin activation and recruitment to depolarized mito-

chondria [12–15,47]. PINK1 phosphorylates parkin

Ubl domain on residue Ser65 to activate parkin [48–50]. Although the phospho-mimetic S65E mutation

stimulates parkin ligase activity in vitro, this mutation

is not able to bypass the PINK1 requirement for mito-

chondrial recruitment in cells and the non-phosphory-

latable S65A mutation is not completely impaired [48].

These observations led to the search for an additional

PINK1 substrate involved in the parkin activation and

the breakthrough discovery that ubiquitin can be phos-

phorylated by PINK1 [51–53]. Three groups concur-

rently observed that PINK1 can phosphorylate

ubiquitin on Ser65, the same position that is phos-

phorylated in the parkin Ubl domain. Further, they

showed that phosphorylated ubiquitin or phospho-

mimetic ubiquitin mutants (S65D/E) directly activate

parkin by enhancing the rate of E2–ubiquitin discharge

[51–53]. Parkin binds strongly to phospho-ubiquitin

with an affinity of 400 nM. Phosphorylation of Ser65

of parkin further increases the affinity 20-fold. In cells,

expression of the non-phosphorylatable S65A ubiqu-

itin delays parkin recruitment to the depolarized mito-

chondria, and mutation of both parkin and ubiquitin

at Ser65 abolishes parkin activation [53]. Further stud-

ies have revealed that PINK1 is not only a ubiquitin

kinase but is also capable of phosphorylating ubiquitin

in ubiquitin chains [25,54,55]. While phospho-ubiquitin

can still become activated by E1 and charged onto E2

enzymes, its activity in ubiquitination assays is E3

dependent [55]. Parkin shows somewhat less activity

with phospho-ubiquitin–E2 than with ubiquitin–E2[51]. Potentially more relevant is the observation that

phosphorylated ubiquitin chains were more resistant to

hydrolysis by 10 out of 12 deubiquitinases tested [55].

One possible explanation is the presence of a minor

conformation of phospho-ubiquitin that was detected

by NMR [55]. The minor conformation, corresponding

to about 30% of molecules, shows a b-strand slippage

that disrupts the Ile44 hydrophobic patch involved in

many ubiquitin interactions.

The structural details of parkin activation are

unknown but most of the evidence is compatible with

a simple two-state system. Inputs that activate parkin

shift the equilibrium between an inactive and active

conformation. There is no evidence of multiple steps



in the pathway: disruption of the RING0–RING2

interface increases ligase activity as does disruption of

the REP–RING1 interaction. The conformational

change between the two states has been suggested to

be a ‘butterfly’ movement in which parkin folds along

the axis between the RING0 and RING1 domains to

bring the two functional sites together [28]. Alterna-

tively, the active conformation could consist of an

ensemble of structures where the E2 binding and cata-

lytic sites transiently interact like beads on a string.

The release of the inhibitory interactions would suffice

to activate the RING1 and RING2 domains to carry

out the ubiquitin ligase activity.

The site of parkin phosphorylation is in close prox-

imity to the REP linker between IBR and RING2

domain in the autoinhibited conformation (Fig. 2B)

and could promote displacement of the REP linker

and Ubl domain to allow E2 binding. The site of

phospho-ubiquitin binding to parkin is also unknown

and it is not clear if the phospho-ubiquitin and phos-

phoUbl binding sites are the same or distinct. In one

of the crystal structures, a pocket formed by three

solvent exposed positively charged residues Lys161,

Arg163 and Lys211 in the RING0 domain was occu-

pied by a sulfate ion, suggesting a potential phos-

phate binding site [30]. While the PD-associated

mutants K161N and K211N fail to recruit to depo-

larized mitochondria [12–14], mutation of the basic

patch did not affect the binding affinity towards

phospho-ubiquitin, suggesting another role for this

pocket [25].

Parkin recruitment to mitochondria

In cultured cells, PINK1/parkin pathways can be acti-

vated by depolarizing mitochondria with uncouplers,

such as carbonyl cyanide m-chlorophenyl hydrazone

(CCCP). Following CCCP treatment, parkin shows a

very robust and complete recruitment to mitochondria

within several hours, which is then followed by the

clearance of mitochondria. Parkin recruitment to mito-

chondria requires PINK1 kinase activity; however,

there is no fixed stoichiometry between PINK1 levels

and parkin recruitment. This suggests that a molecule

other than PINK1 is recognized by parkin. This con-

trasts with the surprising observation that PINK1 arti-

ficially targeted to peroxisomes is sufficient to recruit

and activate parkin on peroxisomes [4]. A further

enigma is the requirement for parkin ubiquitin ligase

activity for its recruitment. Catalytically inactive

mutants of parkin do not show detectable recruitment

following mitochondrial depolarization [12,21].

The solution to this puzzle is now emerging with a

model of the events leading to the recruitment and

activation of parkin by phospho-ubiquitin (Fig. 4). In

the first step, the selective accumulation of PINK1 on

depolarized mitochondria leads to the phosphorylation

of low, basal levels of ubiquitin or parkin present on

mitochondria [25,51–53]. The system exhibits feedfor-

ward control as both ubiquitin and parkin phosphory-

lation are positive effectors of parkin ubiquitin ligase

activity [25,51–53]. Phosphorylation of ubiquitin

chains was recently shown to inhibit the action of

PINK1

Ub

UbUbUbUb

UbUb

UbUb

Phagophore

Mitophagy

PINK1 accumulation & activation

Phospho- ubiquitin

Parkin phosphorylation

Parkin recruitment and activation

Ubiquitination of mitochondrial proteins

Autophagy of mitochondria

Phosphorylation

PINK1

parkin

parkin

parkin

Depolarizedmitochondrion

Depolarizedmitochondrion

PINK1

A B

Fig. 4. Pathway of PINK1 activation of parkin leading to autophagy of depolarized mitochondria. (A) Flowchart of feedforward and feedback

activation of parkin. (B) Schematic of the quality control pathway. PINK1 accumulates on depolarized or damaged mitochondria and

phosphorylates ubiquitin and parkin present on the surface. Activated parkin produces additional ubiquitin chains on the mitochondria, which

in turn are phosphorylated by PINK1 to promote the recruitment of more parkin. The autophagosome forms around the heavily ubiquitinated

mitochondria, which are then eliminated by autophagy.



deubiquitinases [55], which would act as an additional

feedforward mechanism. Full activation of the system

requires a positive feedback loop where activation of

parkin increases the amount of mitochondrially conju-

gated ubiquitin, which is then phosphorylated by

PINK1 to recruit more parkin. The positive feedback

explains the requirement for both PINK1 and parkin

catalytic activities as well as how high levels of parkin

can be recruited and depleted from the cytosol by low,

endogenous levels of PINK1. In agreement with the

model, the requirement for parkin ligase activity can

be bypassed by using overexpressed tetra-ubiquitin

chains artificially targeted to mitochondria [56]. The

requirement for PINK1 activity can be further

bypassed by the use of phospho-mimetic chains for

S65E tetra-ubiquitin [54].

A number of questions remain unanswered. The

relative importance of the feedforward pathways is

unknown as is the order of the initial events. It is pos-

sible, although unlikely, that ubiquitin phosphoryla-

tion precedes chain formation and that parkin

incorporates phospho-ubiquitin directly into chains

[25,51,52]. The relative importance of mono-ubiquitin

and polyubiquitin chains is also unknown. Mono-

ubiquitin tethered to mitochondria is not sufficient to

recruit catalytically inactive parkin which suggests that

ubiquitin chains may play a special role [56].

Parkin/PINK1-mediated mitophagy inneurons

Although parkin recruitment to depolarized mitochon-

dria is a robust phenomenon in diverse mammalian

cell lines, it has been controversial whether mitophagy

is applicable in neurons and in PD pathogenesis. Con-

cerns are that most of these experiments use overex-

pressed parkin and that CCCP treatment leads to

rapid depolarization of the entire mitochondrial net-

work and non-physiological levels of damage to mito-

chondria, which probably is never the case in PD. In

experiments in primary neurons, parkin recruitment to

depolarized mitochondria is modest and only happens

after prolonged CCCP treatment or in the presence of

lysosomal or apoptosis inhibitors or special culture

conditions [57,58]. In part, this could arise from the

fact that most cell lines are glycolytic while neurons

are strictly dependent on oxidative phosphorylation

for mitochondrial ATP production; neuronal mito-

phagy studies may require more physiological methods

for induction of depolarized mitochondria. In a recent

paper, the short mitochondrial ARF protein was

shown to induce mitochondrial depolarization and

parkin/PINK1 autophagy both in cell lines and in

neurons [59]. Moreover, light activated ROS-induced

mitochondrial depolarization was also shown to initi-

ate parkin- and PINK1-dependent mitochondrial deg-

radation by autophagy in axonal mitochondria [60].

This confirms that parkin recruitment and activation

on mitochondria is relevant for PD.

Other functions of parkin

Parkin plays a number of roles outside of the induc-

tion of mitophagy. Parkin ubiquitination of outer

mitochondrial membrane proteins such as mitofusins,

optic atrophy 1 (OPA1) and Miro alters the balance of

fission to fusion and mitochondrial motility, facilitat-

ing the isolation of dysfunctional mitochondria from

the mitochondrial network for mitophagy [61]. Parkin

and PINK1 also work together to repair mildly dam-

aged mitochondria in response to mild oxidative stress

through the formation of mitochondria derived vesicles

(MDVs) enriched in oxidized proteins [62]. These

MDVs carry damaged cargo to lysosomes for degrada-

tion [63]. Complementary to mitophagy, parkin and

PINK1 balance the turnover of mitochondria by pro-

moting the synthesis of new mitochondria [64]. Over-

expressing parkin in proliferating cells and SH-SY5

cells increases mitochondrial transcription through

interaction with TFAM [65,66].

Parkin has been reported to promote cell survival

through various mechanisms although how parkin

becomes activated in these pathways is not always

clear. Parkin has been ascribed as preventing cell

death through proteosomal degradation of several pro-

teins such as parkin interacting substrate (PARIS),

aminoacyl-tRNA synthetase complex-interacting

multifunctional protein-2 (AIMP2) and Fbw7b, a sub-

strate-binding adaptor protein subunit of SCF E3

ubiquitin ligase complex [67–69]. Parkin may activate

prosurvival pathways by increasing nuclear factor jB(NF-jB) signaling [70] or decreasing activation of

c-jun N-terminal kinase [71–73]. A number of recent

papers describe interactions between parkin and Bcl

proteins in the mitochondrial apoptotic pathway

[69,74–77]. Parkin transcription is under the control of

p53, and reportedly mediates the Warburg effect of

p53 on glucose metabolism [78]. Parkin has also been

implicated in cell surface signaling by controlling epi-

dermal growth factor receptor internalization and Akt

signaling by ubiquitination of the endocytic scaffold

protein Eps15 [31]. Parkin is a tumor suppressor and

its inactivation has been reported in various human

cancers. Parkin is deleted in 30% of human tumors

and parkin-deficient mice are more susceptible to

tumorigenesis [78–82].



Parkin also has a key role in pathogen defense

through xenophagy, a pathway related to mitophagy.

In xenophagy, bacteria are marked with ubiquitin

chains that recruit ubiquitin-binding autophagy adap-

tors, leading to autophagosome formation and eventu-

ally fusion with the lysosome. The ubiquitinated

substrates and ligases involved in this pathway are

poorly understood. Genomic studies identified parkin

as a susceptibility factor for the intracellular bacterial

pathogen Mycobacterium leprae [83]. In a recent paper,

parkin has been shown to be required for resistance to

intracellular pathogens such as Mycobacterium tuber-

culosis and Salmonella enterica through an autophagy-

dependent mechanism [84]. The shared ancestry

between mitochondria and bacteria points to a com-

mon mechanism of parkin-mediated autophagy, but

whether PINK1 or a related kinase is required for xe-

nophagy is unknown.

Is parkin a good therapeutic target?

The recent progress in understanding the regulation of

parkin activity may seem appealing for new routes for

treating diseases with mitochondrial dysfunction. Par-

kin displays low basal activity and a small increase in

the activation of parkin could be sufficient to slow the

progression of PD in sporadic forms of the disease

where the wild-type protein is present [41]. Although

simplistic, small molecules that mimic phospho-ubiqu-

itin or disrupt autoinhibitory interactions might

enhance its neuroprotective action. In cultured cells,

mutation of Trp403 or Phe463 speeds recruitment of

parkin to mitochondria in a regulated process that

remains dependent on PINK1 and mitochondrial

depolarization [29]. A small molecule that binds tightly

to the pocket occupied by the amino acid side chains

would be expected to have the same effect. Alterna-

tively, the deubiquitinating (DUB) enzymes USP30

and USP15 were recently found to oppose parkin/

PINK1 in mitophagy, making inhibitors of these

DUBs prime candidates for drug design [85,86]. In

contrast, USP8 promotes parkin-mediated mitophagy

and thus agonists of this DUB could be developed

[26]. While it remains speculative with many chal-

lenges, the quality control pathway mediated by

PINK1 and parkin appears to offer multiple therapeu-

tic targets for the treatment of PD and other diseases

caused by dysfunctional mitochondria.

Acknowledgements

We thank Jean-Franc�ois Trempe and Xinlu Li for

reading the manuscript and constructive comments.

This work was supported by the Canadian Institutes

of Health Research grant MOP-125924 and student-

ships from the Fonds de la Recherche en Sant�e du

Qu�ebec in partnership with la Soci�et�e Parkinson du

Qu�ebec and the NSERC CREATE Training Program

in Bionanomachines.

Author contributions

MS, GK, and KG conceived and wrote the review.

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